(1) Summary of the Invention
The present invention relates to protein vaccines and methods of use thereof for the treatment of Pythium insidiosum infections in humans and lower mammals. Further, the present invention relates to a method for preparing the preferred vaccine for the treatment which contains intracellular and extracellular proteins of Pythium insidiosum. 
(2) Description of Related Art
Infections caused by fungal and parafungal organisms are occurring with increasing frequency in patients with debilitating illnesses such as leukemia and AIDS, as well as those undergoing immunosuppressive therapy. Within this group of organisms are the traditional pathogenic fungi and a long list of newly recognized emerging opportunistic fungal and parafungal organisms. Among the emerging pathogens is the oomycete Pythium insidiosum a fungal-like organism in the Kingdom Kromista, Phylum Pseudofungi. Pythium insidiosum is not only psychologically distinct from members of the Kingdom Fungi, but also differs physiologically. This may explain why anti-fungal drugs do not have any effect on pythiosis.
Pythiosis insidiosi particularly occurs in humans and lower animals in the tropical, subtropical, and temperate areas of the world (Cock, W. A. W., et al., J. Clin. Microbiol. 25:344-349 (1987)). The disease was described in the beginning of the century in equines of tropical and subtropical countries including India and Indonesia as well as the USA. Soon, however, it was evident that the disease not only affected equines but other mammalian species. In lower animals infections of the cutaneous tissues, lymphatic vessels, intestines, lungs, and bones have been found. In humans, a deadly arteritis infection, subcutaneous invasion and keratitis occurs.
The currently available drugs used to treat fungal infections have had little or no effect on Pythium insidiosum. Reports of treatment with either amphotericin B or surgery, commonly used to treat this disease in both humans and lower animals, have indicated that 60% of the patients died of their infections. In cases of arterial invasion in humans, amphotericin B did not eliminate the infection (Rinaldi, M. G., et al., Mycology Observer 9:7 (1989); and Thianprasit, M., Trop Dermathol 4:1-4 (1990)), whereas in surgery the main problem has been to determine how much of the infected tissues has to be removed. Thus, relapses are common in surgically treated patients, who must also endure the pain and distress that such an invasive traumatic procedure inflicts on them.
The curative properties of P. insidiosum possessed curative properties was first noticed when Costarrican equine with pythiosis injected with P. insidiosum antigens, in a skin test, resulted in the cure of some of the horses (Mendoza, L., et al., Equine pythiosis in Costa Rica: report of 39 cases. Mycopathologia 94:123-126 (1986)). Simultaneously, a similar vaccine with curative properties was successfully used in equines with the disease in Australia (Miller, R. I., Aust. Vet. J. 57:377-382 (1981)). These two vaccines have been referred to in the literature as Mendoza's and Miller's vaccines respectively (Newton, J. C., et. al., The Compendium 15:491-493 (1993)). Early reports indicated that the antigens used in the P. insidiosum-vaccine possessed unique characteristics, somewhat similar to the features of those reported in Trichophyton verrucosum (Gudding R., et al., Can. Vet. J. 36:302-306 (1994)) and other immunotherapeutic vaccines (Foster, J. S., et al., Vet. Med. Small Ani. Clin. 71, 920 (1976); Pier, A. C., et al., Equine Practice 15:23-27 (1993)).
Miller's vaccine uses sonicated hyphal antigens (Miller, R. E., Aust. Vet. J. 57:377-382 (1981)), while Mendoza's vaccine is prepared from culture filtrate antigens (Mendoza, L., et al., 94:123-126 (1986)). Both vaccines have cured about 53% of vaccinated horses. Mendoza's vaccine, however, has a longer shelf life and milder side effects (Miller, R. I., et al., J. Am. Vet. Med. Assoc. 182:1227-1229 (1983)). In addition to its immunotherapeutic features Mendoza's vaccine also showed some degree of protection. This protection was later found to be of short duration (Mendoza, L., et al., Mycopathologia 119:89-95 (1992)). In 15 years of use more than 300 equines have been cured. Mendoza's vaccine was proved to be consistent and safe. In spite of this, the vaccine only cured early equine pythiosis, but not chronic cases of this disease (Mendoza, L., et al., Mycopathologia 119:89-95 (1992)). Aside from the fact that the vaccine only cured early equine pythiosis cases, nothing was known about the immunogens involved in its curative properties nor the immune mechanisms that triggered the killing of P. insidiosum's hyphae infected tissues.
In a recent study using SDS-PAGE and Western blot analysis, the presence of three immunodominant hyphal proteins was found to be of interest (Mendoza, L., et al., J. Clin. Microbiol. 30:2980-2983 (1992)). The immunoblotting study revealed that the IgG of sera from horses with active pythiosis recognized most of the proteins of P. insidiosum. However, of all the proteins analyzed, three bands, the 32,000-molecular-weight 32K, 30K, and 28K, were particularly prominent. More significantly was the finding that antibodies against these three proteins persisted for long periods of time in the successfully vaccinated horses.
There is a need for vaccines which cure pythiosis. The need is particularly great where the patient is in the chronic stage of the disease.